The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels, and we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. The phenotype of the Rpe65 knockout mouse is due to disruption of the RPE-based vitamin A visual cycle, metabolizing. Consequently, in the Rpe65 knockout mouse there is overaccumulation of all-trans-retinyl esters and total absence of 11-cis-retinal. The function of RPE65 thus appears to be that of the retinol isomerase, the crucial enzyme in visual pigment chromophore regeneration. We have also continued studies on beta-carotene 15,15'-monooxygenase (BCMO1). BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that BCMO1 and RPE65 share a similar mechanism of action. In the past year we have made the following progress: a) Having established a catalytic role (in conjunction with lecithin:retinol acyltransferase (LRAT)) for RPE65 in the synthesis of 11-cis retinol, and identifying it as the long-sought isomerohydrolase, we are investigating the details of its enzymatic mechanism and interactions with other visual cycle proteins. We investigated the effect of missense mutations in RPE65 associated with Leber congenital amaurosis/early onset blindness as well as mutations in mouse Rpe65 to understand their effects on protein structure and stability. b) Previously, we demonstrated a crucial role in enzymatic activity for histidine and acidic residues in BCMO1 (and RPE65) that we hypothesized to be involved in metal coordination. These observations, in conjunction with the predicted structure of a related bacterial enzyme, Synechocystis apocarotenal oxygenase (ACO) confirms a catalytic role for iron in this family of proteins but other crucial aspects of the mechanism (electron transfer, etc.) remain unknown. In light of the ACO structure we are investigating how BCMO1 and RPE65 have evolved to fulfill their functions utilizing the basic structure of the carotenoid oxygenase family. c) We are generating a panel of hypomorphic knock-in mice in the mouse Rpe65 gene by homologous recombination. It is anticipated that these will provide important insight into the variability of RPE65-deficient phenotypes, in comparison with the extreme case of the knockout. d) The identity of putative factors binding to transcription elements in the RPE65 gene promoter is being sought. We have tested one such putative factor, ZNF-492, a KRAB-zinc finger protein and have shown that it has a moderate effect on RPE65 gene transcription. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Kruppel-associated box-A (KRAB-A) while KRAB-B remains intact, and has 12 C2H2 zinc-fingers in tandem arrangement. In ZNF492, we postulate that absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest up-regulation of Rpe65 gene expression.